The present invention relates to an integrated optical interference method according to the preamble of claim 1.
Optical film waveguides consist of a thin waveguiding film of higher refractive index on a substrate of lower refractive index. Strip waveguides consist of a strip of higher refractive index on a substrate of lower refractive index or inlaid into the surface of said substrate. Also the superstrate with which the film or strip waveguide is covered has to have a lower refractive index than the film or strip waveguide. According to geometrical optics the optical waves are guided in film waveguides by total internal reflection in the plane of the film; in strip waveguides they are additionally also guided transversally. An important special case of the film waveguide is the planar waveguide where the substrate is planar.
According to wave optical or electrodynamical theory, optical waves propagate in waveguides in the form of guided modes which are characterized by their frequency .nu. or their vacuum-wavelength .lambda.=c/.nu.,their polarization, their transverse field distribution, and their phase velocity v.sub.p =c/N. Here c is the velocity of light in vacuum and N the effective refractive index of the mode. In planar waveguides the modes are designated according to their polarization as TE.sub.m -(transverse electric) and TM.sub.m -(transverse magnetic) modes. The mode number m=0,1,2, . . . denotes modes with different transverse field distributions. Also in strip waveguides, modes with different polarizations and transverse field distributions can propagate. The effective refractive indices N depend on the frequency .nu., the polarization, the mode number, and the properties of the waveguide, such as the refractive indices of the substrate, the superstrate, and the waveguiding film or strip, and the latters thickness and thickness and width, respectively. Light of the same frequency .nu. can propagate in a waveguide simultaneously in the form of modes of different polarization, for example, in a planar waveguide as TE.sub.m - and as TM.sub.M -modes with the same mode number m=0,1, . . . . The effective refractive indices N(TE.sub.m) and N(TM.sub.M) differ from each other.
In integrated optical two beam interferometers according to the prior state of the art, as the Michelson- or Mach-Zehnder-interferometer, a guided wave or mode is with a beam splitter divided up into two partial waves 1 and 2, which propagate along different paths and are superimposed by a beam recombiner. Beam splitter and recombiner can in planar waveguides be realized, for example, by gratings, and in the case of strip waveguides by 3dB-couplers. The partial waves 1 and 2 interfere with the phase difference .phi..sub.1 -.phi..sub.2 =(2.pi./.lambda.)[.sub.1 N ds-.sub.2 N ds], where the integral .sub.j N ds with the incremental path element ds is the path integral over the effective refractive index N on the path of the partial wave j=1,2 and the expression in the bracket is the optical path difference. The intensity at the output port of the interferometer is EQU I=I.sub.1 +I.sub.2 +2(I.sub.1 I.sub.2).sup.1/2 cos(.phi..sub.1 -.phi..sub.2). (1)
The intensity can be measured photoelectrically. By counting the interference fringes, i.e., the maxima and minima of the intensity I, the values of the phase difference .phi..sub.1 -.phi..sub.2 can be determined as integers of 2.pi. and of .pi., respectively. PA0 For the phase difference .DELTA..PHI.(L) of the two modes at the end of the measuring section it follows from Eq. (2) EQU .DELTA..PHI.(L)=2.pi.(L/.pi.)[N(TE.sub.0)-N(TM.sub.0)]+.DELTA..PHI.(0), (3)
With an integrated optical interferometer according to the prior state of the art changes of the effective refractive index N can be measured, if either the geometrical path lengths in the two legs of the interferometer are chosen to be of different lengths, or if N is changed in one leg only but remains constant in the other leg. Integrated optical interferometers according to the prior state of the art have the disadvantage of being expensive; their fabrication is complicated since microstructures have to be very precisely produced.
The object of the present invention is to provide an integrated optical interference method for the selective detection of substances in liquid or gaseous samples, and/or for the measurement of changes of the refractive indices of liquid or gaseous samples, and/or of the concentrations of ions, which in spite of its high sensitivity and large measurement range can be realized more easily and cheaply, and requires no waveguides with structures, such as beam splitters, gratings, 3dB-couplers, etc., but only needs a single planar waveguide or a single strip waveguide, and to provide an apparatus which can be directly inserted into the sample to be analysed.
The method and apparatus of the present invention solves the above described problem.
In a planar waveguide the two orthogonally polarized modes are a TE.sub.m -mode and a TM.sub.m,-mode, where the mode numbers m and m' are either identical or different; preferably the two modes are the TE.sub.0 -mode and the TM.sub.0 -mode. The laser light is most easily incoupled into the waveguide through its front face. Also the outcoupling is done most easily through the end or front faces. The incident laser light either has to be linearly polarized under an angle .psi. with respect to the normal on the waveguide, where .psi..noteq.0.degree. and .noteq.90.degree. and preferably is .psi.=45.degree., or it has to be elliptically, preferably circularly, polarized. The outcoupled laser light has two mutually orthogonal polarization components s and p, where the p component, which is linearly polarized in direction of the normal on the waveguide, is generated by the TM-mode, and the s component, which is polarized parallel to the plane of the film, is generated by the TE-mode.
The sample is applied to the surface of the waveguide, at least in a waveguide section called measuring section, and if the surface is coated with a chemically selective sensitive layer, which is called chemo-responsive layer in the following, the sample is applied to this chemo-responsive layer; or a part of the waveguide with the measuring section is inserted into the sample.
The light of the two orthogonal polarization components s and p in the outcoupled light is mutually coherent; between the two components a phase difference .DELTA..PHI.(t) exists which depends on time t if a time dependent change occurs in the sample or when the sample is applied to the measuring section. The phase difference .DELTA..PHI.(t)is measured as a function of time t and therefrom the change of the quantity to be measured is inferred or quantitatively determined. Several particularly suitable methods for the measurement of .DELTA..PHI.(t) are described below.
For the method according to the present invention the following points are essential:
In light propagation through non-birefringent media, such as air or optical elements, such as lenses, the optical paths lengths, i.e., the path integrals over the local refractive index times the incremental path element, are equal for the two orthogonal polarization components. The phase difference between the two polarization components is not changed during their propagation. In the method according to the present invention, this result holds for the light propagation before the waveguide and after the waveguide, i.e., for the paths between the laser and the waveguide and between the waveguide and the photodetector, respectively. Consequently, the phase difference .DELTA..PHI.(t) between the two polarization components is the same everywhere in the outcoupled light. Therefore the interferometer is insensitive against vibrations and temperature changes, which cause optical path length changes. This property is very advantageous because the mechanical construction of the interferometer does not need to have the great stability which some other interferometers in threedimensional optics require.
With polarization optical components, such as phase retardation plates, for example .lambda./4-plates, and/or beam splitters, additional phase differences .PHI..sub.0 between the polarization components can be produced. Such phase differences .PHI..sub.0 which are introduced for the purpose of measuring the phase difference .DELTA..PHI.(t) will be considered in more detail below.
It is very surprising that during the light propagation in the waveguide a phase difference .DELTA..PHI.(t) between the two mutually orthogonal polarized modes arises due to the interaction with the sample. This phase difference .DELTA..PHI.(t) depends on time, if a temporal changes occur in the sample or if the sample is applied to the measuring section. The existence of the phase difference .DELTA..PHI.(t) can be understood in the following way: the light of the modes is guided by total internal reflection in the waveguiding film or strip; however, the field distribution reaches out--in the form of an evanescent wave--into the superstrate, i.e., into the chemo-responsive layer and/or into the sample. The penetration depths of the evanescent fields of the two orthogonally polarized modes are different and, consequently, also the strength of their interaction with the sample.
Quantitatively, this behaviour can be described with the effective refractive indices of the two orthogonally polarized modes, what is explained in more detail for the TE.sub.0 - and the TM.sub.0 -modes of planar waveguides. The effective refractive indices N(TE.sub.0) and N(TM.sub.0) of the two modes are different. Also the effective index changes .DELTA.N(TE.sub.0) and .DELTA.N(TM.sub.0) which arise due to interaction of the modes with the sample are different. Surprisingly it was found, that the difference .DELTA.N.ident..DELTA.N(TE.sub.0)--.DELTA.N(TM.sub.0) can assume rather large values, for example, 30-50% of the value of .DELTA.N(TE.sub.0) itself.
During propagation of the measuring section of length L the phase of a mode is changed by EQU .DELTA..PHI.(L)-.DELTA..PHI.(0)=2.pi.(L/.lambda.)N. (2)
where .DELTA..PHI.(0) is their phase difference at the beginning of the measuring section. If the effective refractive indices change as functions of time t, viz., N(TE.sub.0) by .DELTA.N(t;TE.sub.0) and N(TM.sub.0) by .DELTA.N(t;TM.sub.0), the phase difference .DELTA..PHI.(L) changes with time by EQU .DELTA..PHI.(t)=2.pi.(L/.lambda.).DELTA.N(t), (4)
where EQU .DELTA.N(t)=.DELTA.N(t;TE.sub.0)-N(t;TM.sub.0). (5)
The reasons why the effective refractive indices and, consequently, the phase difference .DELTA..PHI.(t) change with time when the sample changes temporally or is applied to the measuring section will be explained below.
In the case of the planar waveguide, the TE.sub.0 -mode coupled out of the waveguide generates the polarization component s, and the outcoupled TM.sub.0 -mode the polarization component p. Between said two polarization components s and p the phase difference is .DELTA..PHI.(t)+.DELTA..PHI., where .DELTA..PHI. is constant. The phase difference .DELTA..PHI.(t) can be measured with several different methods. Common to all those methods is that the two mutually orthogonally polarized components in the outcoupled light, are superimposed by a polarizer or another polarizing optical component so that they can interfere with each other, and that the resulting intensities I.sub.j (t) are measured in one or several measurements channels j=1, . . . ,M with (M=1,2,3 or 4). If only a single measurement channel is used, the outcoupled light falls through a polarizer onto a photodetector. The transmission direction of the polarizer is orientated to coincide with the bisector of the directions of polarization of the linearly polarized components s and p. The intensity measured by the photodetector is EQU I=I.sub.s +I.sub.p +2(I.sub.s I.sub.p).sup.1/2 cos[.DELTA..PHI.+.DELTA..PHI.(t)], (6)
where I.sub.s and I.sub.p are the intensities that the polarization components s and p would produce separately, .DELTA..PHI. is the constant phase difference, and .DELTA..PHI.(t) is the time dependent phase difference arising from the temporal changes of the quantity to be measured. If maxima and minima of I(t) are counted, then .DELTA..PHI.(t) can be measured with a resolution of .delta.(.DELTA..PHI.)=.pi.. Preferable I.sub.s =I.sub.p is chosen, for maximum contrast of the interference fringes.
The sensitivity of the method according to the present invention is proportional to the length L of the measuring section. An advantage is that the length L can be chosen in a wide range, for example, from a few millimeters to a few centimeters. For a length of the measuring section of, for instance, L=15 mm, at the wavelength of the helium-neon laser .lambda.=633 nm (and at the wavelength .lambda.=750 nm of a laser diode), an effective refractive index change of only .DELTA.N=4.multidot.10.sup.31 5 (and of .DELTA.N=5.multidot.10.sup.-5, respectively) will cause a phase change of .DELTA..PHI.=2.pi.. Depending on the method employed for measuring the phase difference, phase changes .DELTA..PHI.(t) can be measured with a resolution of .delta.(.DELTA..PHI.)=.pi. to .delta.(.DELTA..PHI.)=2.pi./100 and even to .delta.(.DELTA..PHI.)=2.pi./1000. Therefore, in the mentioned example changes in the effective refractive index .DELTA.N of .delta.(.DELTA.N)=2.multidot.10.sup.-5 to .delta.(.DELTA. N)=4.multidot.10.sup.-7, and even to .delta.(.DELTA.N)=4.multidot.10.sup.-8 can be resolved.
The effective refractive index N depends on the refractive index of the medium covering the waveguide and on the thickness of an adlayer adsorbed on the surface of the waveguiding film or strip. Changes in .DELTA.N(t) and, consequently, in the phase difference .DELTA..PHI.(t) arise, if A) the refractive index of the medium covering the measuring section of the waveguiding film or strip changes, or B) substances, i.e., molecules, atoms, or ions are a) deposited on the measuring section of the waveguiding film or strip by unspecific adsorption, chemisorption or binding, i.e., if an adsorbed adlayer is formed, or b) if the waveguiding film or strip is micoporous, are deposited or adsorb in these micropores.
Therefore, with the method according to the present invention the following three measurements can be performed:
1. changes in refractive index of the sample can be measured, in particular for liquid samples. The method according to the present invention is suited for being used as a differential refractometer.
2. the adsorption of substances out of a gaseous or liquid sample can be measured. The method according to the present invention is so sensitive that submonomolecular layers of adsorbed molecules can be detected.
Examples for this are: with waveguiding films of SiO.sub.2 -TiO.sub.2, changes in relative humidity, for instance of the relative humidity of air, can be measured, as water is adsorbed on the surface and is sorbed in the micropores of the SiO.sub.2 -TiO.sub.2 film. The method according to the present invention is suitable as a humidity sensor.
3. specific substances in the sample can be detected selectively. For that purpose a chemo-responsive layer on the surface of the measuring section is required that selectively adsorbs, chemisorbs, or binds the substance to be detected, and thus either changes its refractive index or thickness, or effects the deposition of an adlayer on the chemo-responsive layer. This chemo-responsive layer can also be provided in the micropores of a porous waveguide. A semi-permeable membrane can also be provided in front of the chemo-responsive layer so that only those substances in the sample which diffuse through said membrane can interact with the chemo-responsive layer.
Examples of chemo-responsive layers are the following:
a) the chemo-responsive layer consists of a, for example, monomolecular layer of molecules of an antibody, which are bound, preferably covalently bound, to the waveguiding film or strip. If the antigen or hapten corresponding to that antibody is present in the sample, the antigen or hapten molecules bind to the antibody molecules and thus form an adlayer, the formation of which is detected with the method according to the present invention. The immuno-reaction between the antigen or hapten and the corresponding antibody is highly specific and selective, in particular if monoclonal antibodies are used. Analogously, if the measuring section is coated with antigen or anti-antibody molecules, to those immobilized molecules the corresponding antibody molecules in the sample will bind.
From the literature techniques for production of monoclonal antibodies (MAB's) are known; MAB's against numerous antigens such as bacteria, fungi, viruses, or fragments thereof, and haptens, for example, hormones or toxic substances, have been produced. Therefore the method according to the present invention can be applied in medical diagnostics for the detection of antibodies and antigens in body fluids, in agrodiagnostics for the detection of plant diseases, in the food industry for the detection of bacterial contaminations, and for the detection of toxic substances. Methods for covalent immobilization of antibodies to surfaces, for example to glas and SiO.sub.2 -surfaces, have been described in the literature.
b) From the literature [A. Kindlund and I. Lundstrom, Sensors and Actuators 3,63-77 (1982/83); M. S. Nieuwenhuizen and A. W. Barends, Sensors and Actuators 11,45-62 (1987); Liedberg, Nylander, and Lundstrom, Sensors and Actuators 4,229-304 (1983)] compounds are known, that selectively bind or absorb certain gases and thereby change their refractive index. Examples for this are silicon oils which absorb halogenated hydrocarbons. If those substances are used as chemo-responsive layers, with the method according to the present invention gases, for example, toxic or dangerous gases, can be detected very sensitively.
For the detection of hydrogen, palladium can be deposited on the surface or in the pores of the waveguiding film.
c) With an organophilic chemo-responsive layer, which absorbs or binds hydrocarbons, contaminations such as oil, or Diesel fuel can be detected in water. From the literature [F. K. Kawakara and R. A. Fiutem, Analytica Chimica Acta 151,315 (1983)] it is known that silanes have this organophilic property.
d) The measurement of the concentrations of ions, for example, of H.sup.+ - ions (pH-value) or K.sup.+ -ions, is possible with suitable chemo-responsive layers. Suited for the measurement of pH-values are chemo-responsive layers consisting of indicator dyes, chemisorbed or bound, or embedded in a polymer. Since indicator dyes change their colour with pH-value, they must change their refractive index in wavelength ranges where they exhibit low light absorption, which is a well-known fact following from the dispersion relations. Suitable for the measurement of K.sup.+ -ion concentrations is a chemo-responsive layer consisting, for example, of valinomycin molecules embedded in a polymer, which very selectively bind K.sup.+ -ions.
The dependence of the sensitivity of the method according to the present invention on the properties of the employed waveguide is considered in the following for the case of a planar waveguide and the use of the TE.sub.0 -mode and the TM.sub.o -mode. The sensitivity is proportional to the temporal change of the difference .DELTA.N(t)=.DELTA.N(t;TE.sub.0)-N(t;TM.sub.0) of the effective refractive indices of the two modes, which is induced by the sample. The effective refractive index changes .DELTA.N(t;TE.sub.0) and N(t;TM.sub.0) of the two modes themselves are great, if said modes interact strongly with the sample and with the chemo-responsive layer changed by the sample, respectively. This is the case, if their field is spatially strongly concentrated, i.e., if the effective film thickness d.sub.eff is as small as possible. The effective film thickness d.sub.eff is defined as the sum of the geometrical thickness d of the waveguiding film and the penetration depths of the evanescent fields into the substrate on the one side and into the sample and/or the chemo-responsive layer and the sample on the other side. The thickness d.sub.eff is small if d is small and if the difference n.sub.1 -n.sub.2 of the refractive indices of waveguiding film (n.sub.1) and substrate (n.sub.2) is as great as possible, preferably is n.sub.1 -n.sub.2 &gt;0.25. The film thickness d has to be chosen greater than the cut-off-thickness d.sub.c (TM.sub.0) of the TM.sub.0 -mode, so that both the TE.sub.0 - and the TM.sub.0 -modes can propagate in the waveguide. The range of thicknesses d, in which the sensitivity of the method according to the present invention is high, can be determined without difficulty by the man of the art by simple calculations or a suitable series of experiments in the given case; said range is dependent on the refractive indices n.sub.1 and n.sub.2 and that (n.sub.4) of the sample, and of the refractive indices and thicknesses of the chemo-responsive layer an an adsorbed adlayer. It is advantageous, to choose the thickness d--at least at the measuring section--to be smaller than the cutoff-thickness dc(TE.sub.1) of the TE.sub.1 -mode, so that the guided wave can propagate only as the TE.sub.0 -mode and the TM.sub.0 -mode, and that no modes of higher order m&gt;1 can propagate and potentially can cause disturbances. For waveguiding films of SiO.sub.2 and TiO.sub.2 with n.sub.1 .apprxeq.1.75 on Pyrex glas substrates with n.sub.2 .apprxeq.1.47, the ranges of high sensitivity were found to be: a) in gaseous samples (where n.sub.4 .apprxeq.1) 220 nm&lt;d&lt;420 nm and b) 150 nm&lt;d&lt;390 nm in aqueous samples (where n.sub.4 .apprxeq.1.33), for the measurement of refractive index changes of the sample and of the adsorption of proteins on the waveguide surface. The given values for the lower bound of d correspond to about 5/3 of the cutoff thickness d.sub.c (TM.sub.0) of the TM.sub.0 -mode.
The waveguiding films or strips are preferably made of materials with a high index of refraction n.sub.1, for example, of mixtures of SiO.sub.2 and TiO.sub.2 (n.sub.1 .apprxeq.1.75) or of Si.sub.3 N.sub.4 (n.sub.1 .apprxeq.2.0) on substrates with much smaller refractive index n.sub.2, made preferably of glas (n.sub.1 &lt;1.5) for example, of Pyrex glas. As substrate also a silicon wafer can be used, if the surface of which has been coated with a buffer layer that does not absorb light, preferably consisting of SiO.sub.2, to avoid any attenuation of the guided wave. The waveguiding films or strips can also be fabricated of polyimid (n.sub.1 .apprxeq.1.8) on substrates made of glas or polymer, preferably polymethylmethacrylate (PMMA) or polycarbonate. Films of mixtures of SiO.sub.2 and TiO.sub.2 can, for example, be fabricated by dip-coating from organo-metallic solutions by the sol-gel process; films of Si.sub.3 N.sub.4 by a CVD process.
The present invention is described below, by way of examples in the appended drawings.